Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats. Methods Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups A and B, the SCI model was established with modified Allen’s method and then the rats of groups A and B were administrated by subarachnoid injection of 6 μL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord. Results At different time points, the BBB score of group C was significantly higher than those of groups A and B (P lt; 0.05), and the BBB score of group A was significantly better than that of group B at 14 and 28 days after operation (P lt; 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P lt; 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P lt; 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P lt; 0.05). Conclusion The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.
Objective To investigate the effects of chondroitinase ABC (ChABC) combined with bone marrow mesenchymal stem cells (BMSCs) in repair spinal cord injury of rats. Methods Primary BMSCs were isolated and cultured from the femur and tibia of neonatal Sprague Dawley (SD) rats. The spinal cord injury model was established in 24 adult SD male rats (weighing, 200-230 g), which were randomly divided into control group (group A), BMSCs transplantation group (group B), ChABC injection group (group C), and ChABC and BMSCs transplantation group (group D), 6 rats in each group. At 7 and 14 days after injury, Basso-Beattie-Bresnahan (BBB) score criteria was used to evaluate the hindlimb motor function; at 14 days after injury, the injured spinal cord tissue was perfused and stained by HE for further calculation of the injury area. Immunofluorescence staining were used for observing the expressions of glial fibrillary acidic protein (GFAP)/chondroitin sulfate proteoglycan (CSPG) and GFAP/growth associated protein 43 (GAP43). Results At 7 days after injury, three joints movement of the hindlimbs were recovered in all groups, and no significant difference in the BBB score was found among 4 groups (P gt; 0.05). At 14 days after injury, no load drag was observed in 3 joints of the hindlimbs in groups A, B, and C, but weight-bearing plantar or occasional dorsalis pedis weight-bearing walking was observed in group D with no plantar walking. The BBB score of group D was significantly higher than that of the other 3 groups (P lt; 0.05). HE staining showed that the cavity formed in the damage zone, and there were a large number of macrophages in the cavity and its surrounding, which was wrapped by scar tissue. The damage area of group D was significantly smaller than that of the other 3 groups (P lt; 0.05). At 14 days after injury, the GFAP/CSPG double immunofluorescence staining showed that the astroglial scar damage zone in group D was significantly reduced, and no cavity formation was found. And the fluorescence intensity in groups C and D was significantly lower than that in group B (P lt; 0.05). The GFAP/GAP43 double immunofluorescence staining showed that GAP43-positive fibers passed through the damage zone in group D and the fluorescence intensity in group D was significantly higher than those in groups B and C (P lt; 0.05). Conclusion Inhibition of astrocytes secreting CSPG by ChABC combined with BMSCs transplantation in early injury may promote the regeneration of nerve fibers, and repair spinal cord injury in rats.
Objective To investigate the relationship between the expression of apoptosis-related gene Fas and recovery of neurological function after surgical decompression at different time points in acute spinal cord injury (SCI) rat model by cerclage. Methods A total of 100 13-week-old male Sprague Dawley rats (weighing, 255-376 g) were randomly divided into 4 groups (n=25). The rats only received laminectomy in group A as control; the rats were made the acute SCI models by cerclage in groups B, C, and D. The spinal cord decompression was performed in group B at 8 hours and in group C at 72 hours, no spinal cord decompression in group D. At 1, 3, 7, 14, and 21 days, Basso-Beattie-Bresnahan (BBB) score and inclined plane test were used to evaluate the recovery of neurological function; the neuronal apoptosis level of spinal cord was examined by TUNEL staining; HE staining and immunohistochemical staining were applied to analyze the expressions of Fas. Results The BBB score and inclined plane test score in group A were significantly better than those in groups B, C, and D at different time points (P lt; 0.05); group B was significantly better than groups C and D, and group C than group D at 3, 7, 14, and 21 days (P lt; 0.05). In group A, no bleeding, edema, or necrosis was found. The edema, hemorrhage, and neuron death were observed in spinal cord tissue of groups B, C, and D at 1 day after operation, especially in group D. The degree of cell degeneration in group B was lighter than that in groups C and D at 3 and 7 days after operation; few glial cells and fibroblast proliferation were found at damaged zone in group B at 14 and 21 days, but necrosis and cystic cavity in groups C and D. Fas and TUNEL expression was little in group A at different time points. Fas and TUNEL were expressed in groups B, C, and D; the expressions of Fas and TUNEL reached the maximum at 3 days, and then gradually decreased at 7 and 21 days. The number of positive cells was highest in group D, and the number of positive cells in group B was significantly less than that in groups C and D (P lt; 0.05). Conclusion Early decompression of SCI is beneficial to recovering the neurological function. The Fas signal pathway may play an important role in the apoptosis of neuron and glial cells after SCI.
【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nervegrowth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.
Objective To investigate the effects of allogenic transplantation of acellular muscle bioscaffolds (AMBS) seeded with bone marrow mesenchymal stem cells (BMSCs) on the repair of acute hemi-transection spinal cord injury (SCI) in rats. Methods AMBS were prepared by reformed chemical approach and sterilized by compound cold sterilization; BMSCs were harvested by density gradient centrifugation and cultured with adherent method. The 3rd generation BMSCs labeled by Hoechst 33342 were injected into AMBS to construct the BMSCs-AMBS composite scaffolds; the biocompatibility was observed under scanning electron microscope (SEM) and fluorescence microscope in vitro at 14 days. Forty-eight adult female Sprague Dawley rats were used to build SCI model by hemi-transecting at T9-11 level, then randomly divided into 4 groups (n=12). Defects were repaired with BMSCs-AMBS composite scaffolds, BMSCs, and AMBS in groups A, B, and C, respectively; group D was blank control by injecting PBS. At 1, 2, 3, and 4 weeks after surgery, the functional recovery of the hind limbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. At 4 weeks after surgery, HE staining and immunofluorescent assay were adopted. Results Masson staining and HE staining showed that AMBS was mainly of the collagen fibers in parallel arrange, without muscle fibers. After 14 days of BMSCs and AMBS co-culture, a large number of survival BMSCs labeled by Hoechst 33342 were seen under fluorescence microscope; SEM showed that BMSCs grew and attached to the inner surfaces of AMBS. At 2-4 weeks, the BBB score in group A was significantly higher than that in groups B, C, and D (P lt; 0.05), and it was significantly lower in group D than in the other 3 groups (P lt; 0.05); at 4 weeks, the BBB score in group B was significantly higher than that in group C (t=10.352, P=0.000). HE staining revealed that the area of spinal cord cavity after SCI was markedly smaller in group A than in the other 3 groups; immunofluorescent assay showed that more neurofilament 200 positive fibers and Nestin positive cells were detected in group A than in groups B, C, and D, but glial fibrillary acidic protein (GFAP) positive cells significantly decreased. The integral absorbance (IA) values of GFAP were 733.01 ± 202.04, 926.42 ± 59.46, 1 069.37 ± 33.42, and 1 469.46 ± 160.53 in groups A, B, C, and D, respectively; the IA value of group A was significantly lower than that of groups B, C, and D (P lt; 0.05), and it was significantly higher in group D than in groups A, B, and C (P lt; 0.05). Conclusion With relatively regular internal structures and good biocompatibility, AMBS can inhibit glial scar and enhance the survival, migration, and differentiation of BMSCs, so AMBS is the ideal nature vector for cell transplantation. Co-transplantation of AMBS and BMSCs has synergistic effect in treating SCI, it can promote rat motor function recovery.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
Objective To compare single cell suspension of neural stem cells (NSCs) with neurospheres transplantation for spinal cord injury (SCI) so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley (SD) rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and gl ial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats (weighing 230-250 g) were made the SCI models at T10 level with modified Allen method and randomlydivided into 3 groups (20 rats in each). The injury sites were treated by injecting 5 μL sal ine (group A), 5 μL single cellssuspensions of NSCs at passage 3 (group B), and 5 μL neurospheres cell suspensions at passage 3 (group C). At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 (MAP-2). Results The cells cultured were NSCs by morphological observation and immunofluorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days (P gt; 0.05). BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences (P lt; 0.05). HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days (P gt; 0.05). At 14, 21, and 28 days, the number of MAP-2 positive cells of groups B and C was significantly more than that of group A, and group C was more than group B, showing significant differences (P lt; 0.05). Conclusion Transplantation of neurospheres suspension compared with single cell can significantly promote NSCsto differentiate into neurons and is conducive to recover the lower extremity function after SCI.
Objective Methylprednisolone (MP) is the only active drug for acute spinal cord injury (SCI), but the molecular mechanism is still further studied. To investigate the pathophysiology of SCI and the molecular mechanism of MP in treating SCI. Methods Nine rabbits were randomly divided into 3 groups, weighing (3 100 ± 140) g: sham operation group(group A, n=3), model group (group B, n=3), and drug treatment group (group C, n=3). After laminectomy was performed in3 groups, no treatment was given in group A, and the model of SCI was establ ished with modified Allen’s fall ing strike method in groups B and C at L4; then high-dose MP equivalent with human dose was adopted in group C at 2 hours after SCI and the normal sal ine in group B. All rabbits were sacrificed at 8 hours after SCI, and then the spinal cord tissues about 8 mm long which included the injuried site were obtained. Total RNA was isolated with Trizol one-step method to examine the gene expression profile by using Ogl io technologies with standard operating procedures and qual ity control as recently described respectively. GeneSpring11.0 analyzer software was used to filter potential candidate genes for statistical significance using Welch’s t test, and only genes with P lt; 0.05 and fold change (FC) ≥ 2 were retained for further analysis. Some differentially expressed genes were also verified by RT-PCR to ensure the rel iabil ity of microarray results. Results The SCI model was set up and the samples of spinal cord tissues were acquired successfully at 8 hours after SCI. The qual ify of total RNA from each group met the requirement for the microarray examination and data analysis. These differentially expressed genes involved inflammation, immunity, ion transportation, transcription factors, and so on. The results of genes IL-1α, IL-1β, and defensin 4 (NP-4) by RTPCR were consistent with that of gene-chips. The immuno-related genes included NP-3, NP-4, corticostatin 6, CAP-18, and antimicrobial peptide, which displayed obvious differential expression. Conclusion High-dose MP has protective effects on nervous function by the immunity mechanism, and the main effector may be neutrophil.